| Background and Objective:Obesity is a chronic metabolic disease with complex etiology,which can cause hypertension,type II diabetes,nonalcoholic fatty liver disease,etc.The incidence rate of obesity is increasing year by year,which has become a major problem of world public health and seriously affects human health and quality of life.The latest clinical data shows that obesity exacerbates the severe risk of COVID-19 infection.The probability of obese patients entering the ICU after COVID-19 infection increases by 74%,and the probability of death increases by 48%.Therefore,prevention and treatment of obesity has important practical significance.Aquaporin（AQP）is a family of homologous tetramer proteins that participate in the transport of water and other solutes on the cell membrane.Aquaporin 8（AQP8）is a member of the aquaporin family.It is highly expressed in the capillaries of hepatocytes and participates in the process of bile secretion by regulating the permeability of water.Bile acid（BA）is an important component of bile.and bile acid synthesis in liver is an important pathway of cholesterol catabolism.It is known that bile acids help to emulsify fat and promote intestinal absorption of lipids.Recent studies have shown that bile acids play an important role in controlling glycolipid metabolism and body energy balance.According to our previous experimental results,the body weight and body fat rate of AQP8-/-mice were significantly higher than those of wild-type mice after high-fat diet,and there was no difference in normal diet.We speculate that the changes of bile acid synthesis-related enzymes and bile acid transporters in AQP8-/-mice after high-fat diet may be one of the reasons for the aggravation of obesity.Therefore,this experiment focuses on bile acid to study the mechanism of obesity aggravation in AQP8 gene knockout mice after high-fat diet,and to explore whether AQP8-/-aggravating obesity under the induction of high-fat diet plays a role through the synthesis and transport pathway of bile acid,thus clarifying the relationship between bile acid and obesity.Methods:Four week-old SPF grade wild-type C57BL/6J mice and AQP8-/-C57BL/6J mice were fed with high-fat diet for 8 weeks as wild-type high-fat diet for 8 weeks（C57HFD8W）and AQP8-/-high-fat diet for 8 weeks(AQP8-/-HFD8W).The weight of mice was measured weekly and the weight growth curve was recorded.Body fat rate was measured one day before death.After taking blood from the canthus vein,the mice were killed by dislocation,and the liver tissues were collected.The levels of total cholesterol（TC）,triglyceride（TG）,high-density lipoprotein（HDL）,and low-density lipoprotein（LDL）were measured with an enzyme marker.The expression of bile acid nuclear receptors,bile acid synthesis-related enzymes,bile acid related transporters,inflammatory factors and PI3K-AKT signal pathway molecules were detected at the m RNA and protein levels by real-time fluorescence quantitative analysis（q PCR）and Western blot.Result:1.In AQP8-/-mice,obesity was exacerbated and blood insulin was significantly increased by a high-fat diet.Compared with the C57HFD8W group,the mice in the AQP8-/-HFD8W group gained significantly more weight（P<0.01）and had a significantly higher body fat ratio（P<0.01）.Intrahepatic TC,TG,and LDL contents were significantly increased（P<0.01,P<0.05,and P<0.05,respectively）.HDL content,although higher than in C57HFD8W mice,was not statistically significant.In addition,fasting glucose and serum insulin levels were similarly significantly elevated（P<0.01 and P<0.001,respectively）.The results showed that after high-fat feeding,the AQP8-/-HFD8W group mice developed exacerbated obesity and developed hyperinsulinemia.2.Increased intrahepatic fat synthesis and decreased lipolysis after high-fat feeding in AQP8-/-mice.（1）The protein expression level of sterol regulatory element-binding protein-1c（SREBP-1c）in the liver of AQP8-/-HFD8W mice was significantly up-regulated（P<0.01）,The m RNA and protein expression level of fatty acid synthase（FAS）,the downstream molecule of SREBP-1c,was significantly up-regulated（P<0.001 and P<0.01）.（2）Peroxisome proliferator-activated receptorα（PPARα）in liver of AQP8-/-HFD8W mice The protein expression level was significantly decreased（P<0.01）,PPARαThe protein expression level of the downstream molecule carnitine palmitoyl transferase1（CPT1）was also down-regulated（P<0.05）.The above results showed that the synthesis of fat in the liver of AQP8-/-HFD8W mice increased and the decomposition decreased.3.FXR/SHP expression is decreased after high-fat feeding in AQP8-/-miceThe protein expression levels of farnesoid X receptor（FXR）in the liver of AQP8-/-HFD8W mice were significantly downregulated（P<0.01）and that of small heterodimer（SHP）,a downstream molecule of FXR,was significantly downregulated（P<0.001）.Our results suggest that FXR acts as a nuclear receptor for bile acids and that impairment of the FXR/SHP signaling pathway promotes hepatic lipid synthesis.4.Increased bile acid synthesis classical pathway and decreased alternative pathway after high fat feeding in AQP8-/-mice.Cholesterol-7,the rate limiting enzyme of the classical pathway involved in intrahepatic bile acid synthesis,was increased in AQP8-/-HFD8W mice cholesterol 7α-Hydroxylase,（CYP7A1）and sterol 12α-Hydroxylase（CYP8B1）showed a significant increase in protein expression（P<0.05 and P<0.01,respectively）,whereas sterol,the rate limiting enzyme of the alternative pathway,sterol 27-Hydroxylase（CYP27A1）and oxysterol 7α-Hydroxylase,（CYP7B1）m RNA expression levels were significantly downregulated（P<0.001 and P<0.01）.The results indicated that the bile acid synthesis pathway was altered in AQP8-/-HFD8W mice.5.Intrahepatic bile acid secretion and reabsorption are significantly decreased after high-fat feeding in AQP8-/-miceThe m RNA and protein expression levels of bile salt export pump（BSEP）,a transporter of bile acid secreted from liver to bile,were significantly decreased in AQP8-/-HFD8W group（P<0.001 and P<0.05）,and the m RNA expression level of multidrug resistance associated protein 2（MRP2）was decreased（P<0.001）.The m RNA and protein expression levels of Na+taurocholate cotransporting polypeptide（NTCP）,a transporter of bile acid reabsorption in liver,were significantly decreased（P<0.001 and P<0.01）,and the m RNA expression level of organic anion transporting polypeptide 1（OATP1）was decreased（P<0.001）.Our results showed that AQP8-/-HFD8W group mice had decreased bile enterohepatic circulation function after high-fat feeding.6.Reduced PI3K-Akt expression after high fat feeding in AQP8-/-miceThe protein expression levels of p-PI3K,PI3K were down regulated in AQP8-/-HFD8W mice（P<0.05）,and the protein expression levels of p-AKT,AKT were similarly down regulated（P<0.01）.Our results showed that the expression of the phosphatidyl inositol 3-kinase（PI3K）/protein kinase B（PKB/AKT）signaling pathway was downregulated after high-fat feeding in AQP8-/-mice,resulting in decreased glycogen anabolism and increased insulin resistance.7.SIRT1 expression is decreased after high fat feeding in AQP8-/-mice,while NF-κB and TNFαare elevated expressions.The protein expression levels of recombinant sirtuin 1（SIRT1）in the liver of AQP8-/-HFD8W mice were lower than those in wild-type C57 mice（P<0.01）,as were nuclear factor kappa-B（NF-κB）and tumour necrosis factor-α（TNFα）than wild-type C57 mice（P<0.05）.Our results showed that AQP8-/-mice had decreased hepatic anti-inflammatory factors,increased pro-inflammatory factors,and aggravated inflammatory responses.Conclusions:1.AQP8-/-mice have reduced intrahepatic FXR/SHP expression,increased fat synthesis and reduced lipolysis after high-fat feeding for 8 weeks,resulting in an exacerbated obesity.Blood insulin is increased and PI3K-AKT expressions are decreased,resulting in a marked insulin resistance.2.AQP8-/-mice decrease intrahepatic FXR/SHP expression,show increased bile acid synthesis by classical pathway,and decreased by alternative pathway after high-fat feeding for 8 weeks.3.The secretion and reabsorption of bile acids are significantly decreased after high-fat feeding for 8 weeks in AQP8-/-mice.4.SIRT1 expression is decreased and expressions of NF-κB and TNFαare elevated after high-fat feeding for 8 weeks in AQP8-/-mice,representing aggravated intrahepatic inflammatory response. |
